In the previous grant period, we found that both estrogen receptor (ER) subtypes, ERa and ERb, are expressed in the glomerulus. ERa the predominant subtype, mediates estrogen-induced expression of matrix metalloproteinases (MMP). Glomeruli and mesangial cells isolated from young ROP (glomerulosclerosis-prone) mice have lower levels of ER expression than those from young B6 (sclerosis-resistant) mice. Since renal disease is more common after menopause, we will focus this continuation proposal on renal disease in normal aging women. We chose B6 female mice because they develop severe glomerulosclerosis in anestrus. This fact provides support for the hypotheses of this application that estrogen and/or estrogen receptor status play a major role in glomerulosclerosis in aging women. We will determine whether ER expression and responses decline in B6 glomeruli in anestrus. We will examine the specific role that each ER subtype plays in glomerulosclerosis using female ER knockout (ERKO) B6 mice in which ERa, ERb, or both ERs are eliminated by gene targeting. Since TGF-bl is a mediator that plays a major role in glomerulosclerosis, we will examine the interactions of the ER and the TGF-bl signaling pathways. We will examine mesangial cells as well as podocytes. We propose the following: Specific Aim I. Determine the effects of anestrus and estrogen supplementation on estrogen responsiveness in glomeruli:(a) Determine whether glomerular ER expression declines in anestrus; (b) Determine if the decline in ER number is reversible and the time when estrogen replacement should be initiated. Specific Aim II. Determine the levels of the ER subtypes response to estradiol, and regulation in mesangial cells and podocytes(a) Mesangial cells isolated from B6 mice in anestrus; (b) Podocytes isolated from female B6 mice before and after anestrus;(c) Determine the means of regulation of ER expression in mesangial cells and podocytes in anestrus. Specific Aim II1. Determine the ER subtype(s) involved in the development of glomerulosclerosis in B6 mice in which ERa[aERKO], ERb [bERKO], or both ERs [abERKO] are absent)(a) Measure albumin excretion rate and serum creatinine; (b) Examine glomerular changes, including cell turnover, histology, morphometry, and immunochemistry; (c) Examine the mRNA expression of ER subtypes, TGF-bl, type IV collagen, laminin and tenascin in isolated (d) glomeruli, in mesangial cells, and in podocytes. Specific Aim IV. Examine the interactions between ER subtypes and TGF-bl in ERKO and anestrus mice.(a) In vivo: Determine if components of the TGF-bl signaling cascade are altered in the absence of ERa and/orERb in glomeruli of ERKO mice (those with significant albuminuria) and 30 mo intact B6 mice; (b) In vitro: Determine the levels and interactions between the TGF-bl signaling cascade and ER(s) in microdissected glomeruli, in mesangial cells and in3odocytes of ERKO, wild type ERKO, and 30 mo mice.